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. 2020 May 18;11:2464. doi: 10.1038/s41467-020-16315-4

Fig. 3. Postsynaptic targeting of SynTagMA using a PSD95 intrabody.

Fig. 3

a CA1 neurons expressing the unregulated construct: a fusion protein of PSD95 fibronectin intrabody (PSD95.FingR) and CaMPARI2_F391W_L398V (without NES or epitope tags). Scale bars: 20 µm, 2 µm. b CA1 neurons expressing PSD95.FingR-CaMPARI2 with a zinc finger binding sequence (ZF BS) added upstream of the promoter and a zinc finger (ZF) fused to a transcriptional repressor domain (KRAB(A)) and mCerulean as a cytosolic filler (840 nm). Scale bars 12 µm (left panel) and 2 µm (center and right panels). c Postsynaptically targeted SynTagMA (postSynTagMA). As in b, with the ZF-KRAB(A) but no upstream ZF-BS. Note that postSynTagMA is still enriched in spines and the nucleus, leaving the cytoplasm almost free of SynTagMA. Scale bars: upper 20 µm, lower 2 µm. d The unregulated construct is expressed at high levels, leading to near-identical concentrations in dendrites and spines. For individual spines (circles), the spine-to-dendrite green fluorescence ratio was similar to the mCerulean spine-to-dendrite ratio (n = 348 spines, 4 neurons). e The construct with autoregulatory elements shuts off its own production, resulting in strong enrichment in spines (n = 367 spines, 3 neurons). f The construct without zinc finger binding sequence (postSynTagMA) is also auto-regulated, showing equally strong enrichment in spines (n = 179 spines, 3 neurons). All images are two-photon (2P) maximum intensity projections. In d, e and f, the black line is the linear fit to data points and the dotted line is the unity line.